Transformants expressing the codon-optimized luciferase gene from G. princeps : phenotype, detection of luciferase activity, and Southern blot analysis. (A) Phenotypes of transformants T-PL2 and T-PL3, which were co-transformed with the selectable marker plasmid pPmr3 and the non-selectable plasmid pPsaD-GLuc. (B) Luciferase assay of wild type and transformants T-PL2 and T-PL3 (at 20°C). Control: medium only. Upper row: standard photo showing the assay setup. Middle row: photo without any extraneous light after addition of the coelenterazine substrate. Lower row: exposure to a light-sensitive film for 30 s. (C) and (D): Southern blot analysis of genomic DNA from transformants T-PL2 and T-PL3 and from the parent wild type strain. The blot in (C) was probed using a fragment of the aphVIII gene, the blot in (D) was probed using a fragment of the luciferase (luc) gene. B, BamHI; E, EcoRI; H, HindIII; P, PstI; S, SalI; Sc, SacI.